Abstract Opioid and catecholamine receptors are key regulators of neurophysiology and behavior, and are important targets of therapeutic and abused drugs. These receptors are fundamentally regulated by endocytosis and specific membrane trafficking events in the endocytic pathway. Endocytic membrane trafficking has long been recognized to influence adaptation or maladaptation of the endogenous opioid and catecholamine systems to chronic or repeated drug administration. It is now evident that endocytic trafficking of receptors also impacts the acute response and may thereby differentiate the actions of therapeutically and addiction -relevant drugs over a wide time frame. In the previous funding period we defined much of the biochemical machinery determining the endocytic regulation of opioid receptors, and established a effects on both long-term and acute signaling. We also developed a new class of biosensors to probe receptor and G protein activation in intact cells, and discovered using these sensors a discrete endosome-localized site of receptor signaling by coupling to G protein. The proposed studies build on progress made in the previous funding period to elucidate a fundamentally new mechanism of opioid and adrenergic receptor sorting in the endocytic pathway, a new mechanism for controlling the initial endocytosis of receptors, a discrete mode of receptor insertion to the surface of dendrites, and a previously unanticipated role of the endocytic machinery in shaping the acute signaling response. We propose to (1) Define how opioid and adrenergic receptors differentially engage the retromer.; (2) Determine if the MOR cytoplasmic tail engages a discrete inhibitory protein at clathrin-coated pits; (3) Define function of the retromer-dependent recycling machinery in relevant neurons; and (4) Pioneer nanobody-based biosensors as probes of G protein activation on endosomes.